1. Fluorescent dye method (SYBR Green)
The principle is: to add excess fluorescent dye to the PCR reaction system, during the process of DNA amplification, the fluorescent dye is specifically incorporated into the double-strand DNA to emit a fluorescent signal. As the reaction progresses, the fluorescence intensity gradually increases, and can Measurement. If you were to amplify your target sample for 40 cycles, the fluorescence detected at the end of the last cycle would be much stronger than at the 10th cycle.
Advantage:
(1). Low cost, suitable for large-scale experiments.
(2). It is very time-saving in the experimental design stage because it only requires a suitable primer design.
Shortcoming:
(1). The specificity is not very high. The dye can intercalate into any double-stranded DNA, including primer dimers and nonspecific products.
(2). If primer dimers are present or the product is contaminated, the results obtained will be unreliable.
(3). Easier to generate non-specific fluorescence with low-abundance targets.
Additional time is required for analysis of results.
2. Oligonucleotide probe (Taqman)
This method involves the use of fluorescently labeled oligonucleotides (short DNA molecules), and the probes are usually labeled at both the 5′ and 3′ ends. (You may need Oligonucleotides service)
There is a reporter group at the 5′ end of the fluorescent probe, and a quencher group is designed at the 3′ end. When the reporter group is close to the quencher group, no fluorescent signal will be detected. During the RT-qPCR reaction, the two groups of the oligonucleotide are separated, and the fluorescent signal can be detected and synchronized with the PCR product.
Fluorescence detection using this method relies on two processes:
(1) Combination of primers and target sequences
(2) Binding of the probe to the downstream complementary sequence of the primer.
Advantage:
(1). It is more likely to amplify only the desired product due to the specificity of the combination of primers and probes.
(2). No dissociation curve is required, and fluorescence can only be detected when the probe binds to the correct target sequence.
(3). Due to the specificity, the data is more reliable.
(4). Save time during data analysis.
Shortcoming:
(1). High cost when large-scale experiments are required.
(2). It takes longer to design experiments because good primers and probes are needed.
In general, the probe method can increase the specificity of the signal collected by the reaction through the probe, and the signal can only be collected when the fragment bound by the probe is amplified. The probe method can use multiple system reactions and can predict and perform reactions in advance The disadvantage of optimizing the conditions is that the probe needs to be synthesized and the cost is high.
The dye method is economical and can do melting curves and analyze the TM value of all PCR products. The disadvantage is that the specificity is not as good as the probe method. Both fluorescence detection methods are very effective, but for your specific experiment, you can choose the appropriate method.
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Related Reading:
Classification and application of fluorescent dyes
The use of fluorescent dyes and how to judge the brightness?
Principle and Analysis Method of Fluorescence Immunoassay (FIA)